PROJECT SUMMARY/ABSTRACT Mitochondrial diseases are a heterogenous class of metabolic disorders affecting 1:5000 people with poor prognosis and no effective treatment options. The underlying mechanisms of organ-specific phenotypes in mitochondrial disease are still unknown. In patient-derived cell lines, glucose is re-routed towards lactate production, and lactic acidosis is a feature of mitochondrial diseases. Metabolic reprogramming of one carbon metabolism, specifically an increase in transsulfuration genes has been identified; however, these results have not been investigated in vivo. This proposal aims to understand how transsulfuration serves as a compensatory mechanism in mitochondrial hepatopathy. To accomplish this, stable isotope tracing mass spectrometry and mouse models of mitochondrial hepatopathy will be utilized. The specific aims of this proposal intend to (1) identify if transsulfuration is remodeled in hepatic mitochondrial dysfunction, (2) test how transsulfuration activity affects liver metabolism and redox status, and (3) determine how cysteine-stimulated transsulfuration promotes glucose metabolism. The findings of this proposal will provide insights into the metabolic reprogramming of a large class of metabolic diseases.